Targeting Poly(ADP)ribose polymerase in BCR/ABL1-positive cells

BCR/ABL1 causes dysregulated cell proliferation and is responsible for chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1-ALL). In addition to the deregulatory effects of its kinase activity on cell proliferation, BCR/ABL1 induces genomic instability by downregulating BRCA1. PARP inhibitors (PARPi) effectively induce cell death in BRCA-defective cells. Therefore, PARPi are expected to inhibit growth of CML and Ph1-ALL cells showing downregulated expression of BRCA1. Here, we show that PARPi effectively induced cell death in BCR/ABL1 positive cells and suppressed colony forming activity. Prevention of BCR/ABL1-mediated leukemogenesis by PARP inhibition was tested in two in vivo models: wild-type mice that had undergone hematopoietic cell transplantation with BCR/ABL1-transduced cells, and a genetic model constructed by crossing Parp1 knockout mice with BCR/ABL1 transgenic mice. The results showed that a PARPi, olaparib, attenuates BCR/ABL1-mediated leukemogenesis. One possible mechanism underlying PARPi-dependent inhibition of leukemogenesis is increased interferon signaling via activation of the cGAS/STING pathway. This is compatible with the use of interferon as a first-line therapy for CML. Because tyrosine kinase inhibitor (TKI) monotherapy does not completely eradicate leukemic cells in all patients, combined use of PARPi and a TKI is an attractive option that may eradicate CML stem cells.


Results
Effect of olaparib on leukemia cells. Expression of BRCA1 by bone marrow-derived cells from BCR/ ABL1 transgenic (Tg) mice was lower than that by cells from wild-type (WT) mice (Fig. 1a). BRCA1 is a key molecule involved in the HRR pathway. Therefore, we examined the effect of BCR/ABL1 expression on HRR activity. As expected, HRR activity was downregulated upon expression of BCR/ABL1 (Fig. 1b). These results suggest that BCR/ABL1-expressing cells exhibit homologous recombination defects (HRD). Next, bone marrow-derived mononuclear cells (MNC) from WT and BCR/ABL1 Tg mice were exposed to the PARPi olaparib in vitro, and cell death was analyzed by Annexin V/propidium iodide staining. Olaparib induced cell death in both WT and BCR/ABL1 Tg mouse-derived MNCs in a dose-and time-dependent manner. BCR/ABL1 Tg mouse-derived MNCs were more sensitive to olaparib than those from WT mice (Fig. 1c,d).
Olaparib prevents transformation activity by BCR/ABL1. It would be interesting to ascertain whether olaparib prevents transformation, or whether PARP is required for transformation by BCR/ABL1.  www.nature.com/scientificreports/ Therefore, we examined BCR/ABL1-mediated transformation activity in Rat-1 cells 12 . Rat-1 cells were either mock-infected or infected with BCR/ABL1 (Supplemental Fig. 1a), and colony formation activity was monitored in the presence or absence of olaparib. Treatment with olaparib reduced the colony transformation activity of BCR/ABL1 (Supplemental Fig. 1b,c).
Olaparib reduces the potential of BCR/ABL1-expressing cells to repopulate HSCs. Next, we performed colony assays to examine the repopulation activity of HSCs in order to analyze the effect of PARP inhibition. The colony-forming activity of HSCs from WT and BCR/ABL1 Tg mice was examined sequentially after replating under continuous olaparib exposure. Intriguingly, colony-forming activity by BCR/ABL1expressing HSCs was abolished after the third replating, whereas wild-type HSCs retained this activity (Fig. 2a). We hypothesized that genomic instability reduces the HSC-repopulating potential of BCR/ABL1-expressing cells. Usually, HSCs arrest at G0 and only enter the cell cycle if they are stimulated. Also, CML progenitors demonstrate increased susceptibility to repeated cycles of chromosome damage, repair, and damage via a breakage-fusion-bridge (BFB) mechanism 13 . Therefore, we examined BFB generation under conditions of cytokine stimulation. Even though cells were stimulated with cytokines, the number of wild-type cells was not different from that of BCR/ABL1-expressing cells. As expected, as cells progressed through the cell cycle, the number of BFBs in the Parp1 -/and Parp1 +/+ BA Tg/− HSC population increased moderately, whereas the number in the Parp1 −/− BA Tg/− HSC population was markedly higher than that in the Parp1 +/+ , Parp1 −/− , and Parp1 +/+ BA Tg/− HSC populations ( Fig. 2b and Supplemental Fig. 1d).
Next, to gain insight into how olaparib affects stem cell maintenance, we compared differentially expressed genes between DMSO-treated and olaparib-treated cells using RNA sequencing-based transcriptome analysis. www.nature.com/scientificreports/ When we focused on highly expressed genes in olaparib -treated cells, we identified the genes associated with the TP53 signaling pathway (Table S1 and Supplemental Fig. 2a). Conversely, a focus on downregulated genes in olaparib-treated cells identified genes involved in oxphosphorylation (OXPHOS) (Table S1 and Supplemental Fig. 2b).

Activation of the cGAS/STING pathway in BCR/ABL1-expressing cells. PARPi have a broad range
of biological effects 21 that may have caused the observed reduction in survival of BCR/ABL1-positive cells. The cGAS/STING pathway, which is responsible for de novo synthesis of antiviral type I interferons (IFNs) and their related gene products, is triggered by cytosolic DNA to induce antitumor immune responses 22 . Accumulation of DNA damage following PARP inhibition leads to leakage of damaged double-stranded DNA into the cell cytoplasm, which activates innate immune signaling through the cGAS-STING pathway, leading to increased expression and release of type I IFN 17,23,24 . IFN was once the standard frontline treatment for CML because its pleiotropic mechanism of action includes immune activation and specific targeting of CML stem cells 25 . Therefore, based on the hypothesis that cGAS/STING pathway-mediated activation of the IFN machinery exerts cytotoxic effects on CML LSC, we investigated the effect of olaparib on activation of the cGAS/STING pathway. As expected, olaparib induced cGAS-bound micronuclei (Fig. 3a,b) and TBK1 phosphorylation, which is crucial for STNG activation (Fig. 3c,d). Furthermore, we observed increased expression of IFN-α and CCL5 mRNA (Fig. 3e, and Supplemental Fig. 3a,b). RNA sequencing also revealed upregulation of IFN-responsive genes (Fig. 3f).
Olaparib inhibits BCR/ABL1-dependent leukemia in vivo. Next, we evaluated the effects of PARP inhibition in BCR/ABL1-expressing cells using an in vivo model of hematopoietic cell transplantation. Mouse HSCs were infected with a BCR/ABL1-expressing retrovirus and then transplanted into lethally irradiated mice. Starting at 1 day post-transplantation, mice received an oral 100 mg/kg olaparib (five times per week) or vehicle. Death from BCR/ABL1-mediated leukemia was observed in sham-treated mice at 1 month post-transplantation. All of the mice that received vehicle died within 6 months. However, none of the olaparib-treated mice developed leukemia, and all survived for 6 months (Fig. 4a). After all sham treated mice had died, olaparib administration to the other mice was terminated, and their survival was monitored for about 12 months posttransplantation. Three out of six mice died after termination of olaparib. Overall survival was 50%. Thus, olaparib extends survival significantly (p = 0.0005).

Parp1 knockout increases survival of BCR/ABL1 transgenic mice.
To further confirm the role of PARP inhibition in preventing BCR/ABL1-mediated leukemogenesis, we used a genetic approach. Instead of inhibiting PARP using an inhibitor, we crossed Parp1 knockout mice with BCR/ABL1 Tg mice and examined leukemia development and death. As shown previously, leukemia in BCR/ABL1 Tg mice developed 6 months after birth, after which time the mice started to die 18 . Although there was no difference in white blood cell counts between Parp1 wild-type (Parp1 +/+ BA Tg/− ) and Parp1 knockout BCR/ABL1 Tg mice (Parp1 −/− BA Tg/− ) (Supplemental Fig. 4), we found it interesting that leukemia development was delayed in Parp1 −/− BA Tg/− ; these mice survived longer than Parp1 +/+ BA Tg/− mice (Fig. 4b).

Discussion
Targeting BRCA1/2deficient tumors with PARPi is a standard therapeutic option for HBOC. Currently, PARPi are being developed to target not only BRCA1/2defective HBOC, but also other types of cancer that harbor HRD 26 . Therefore, the genomic instability mediated by BCR/ABL1-mediated downregulation of BRCA1 expression in CML and Ph1-ALL is an attractive candidate for targeted therapy with PARPi. Here, a murine transplantation model yielded data supporting the potential of PARPi for the treatment of BCR/ABL1-positive leukemia. This result was also supported by results obtained using genetic models constructed by crossing Parp1 knockout mice with BCR/ABL1 transgenic mice, and by previous reports 27,28 . PARPi exert their function against multiple PARP family proteins. Among them, olaparib is a potent inhibitor of PARP1 and PARP2. Here, we assessed only Parp1 knockout mice. Although 29 . This difference may be due to the supporting effects of PARP2 on PARP1. We and others showed previously that PARPi have different cytotoxic effects depending on the BCR/ABL1-positive leukemic cell line [30][31][32] ; however, these studies did not characterize differences between the cell lines 30,33 . By contrast, PARPi exerts a cytotoxic effect against MNCs and HSCs from BCR/ABL1 Tg mice. Therefore, we hypothesized that these conflicting results can be explained by different genetic changes in different cells. Accumulation of multiple genetic alterations and disruption of cell signaling pathways play crucial roles in leukemic transformation. Nontransformed cells from BCR/ABL1 Tg mice carry relatively simple genetic alterations, i.e., those that affect only BCR/ABL1 expression, which mimics the chronic phase of CML.
The results of our in vitro repopulation colony assay suggest that PARPi attenuates HSC homeostasis in BCR/ ABL1-positive cells. HSCs localize in hypoxic environments and so are not able to generate ATP using oxygenconsuming mitochondrial OXPHOS. Therefore, they use the glycolytic system predominantly. Whereas CML LSCs show higher TCA cycle flux and mitochondrial respiration than their normal HSCs counterparts, LSCs rely more on OXPHOS, in keeping with their glycolytic metabolic profile 34 . Therefore, downregulation of OXPHOS genes via PARP inhibition may contribute to eradication of BCR/ABL1-positive LSCs. LSCs accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage 35 . Thus, HSCs become exhausted. HSCs from Atm/Foxo knockout mice, or from other mouse models defective in DNA repair, exhibit premature exhaustion due to accumulation of ROS and/or DNA damage 36  www.nature.com/scientificreports/ embryos 29 . Farees et al. report that Parp2 −/− mice exposed to sublethal doses of irradiation exhibit bone marrow failure, which correlates with reduced long-term repopulation of irradiated Parp2 −/− HSCs under competitive conditions 38 . In addition, Li et al. reported that activation of PARP1 by salidroside protects quiescent HSCs from oxidative stress-induced cycling and self-renewal defects, both of which are abrogated by genetic ablation or pharmacologic inhibition of PARP1 39 . Expression of BCR/ABL1 augments DNA damage and/or increases ROS production in HSCs. Therefore, under conditions of PARP knockout or PARP inhibition, these cells may easily become exhausted. Another explanation is that HSCs in vivo are in a quiescent state and only proliferate when stimulated. Here, we found no significant difference in the amount of DNA damage in HSCs from Parp1 −/− BA Tg/− and Parp1 +/+ BA Tg/− mice; however, we observed increased genomic instability in proliferating HSCs from Parp1 −/− BA Tg/− mice. Thus, PARP inhibition may accelerate exhaustion of replicating BCR/ABL1positive HSCs.
Historically, IFN has been used as a first-line therapy for patients with chronic-phase CML who are not eligible for allogeneic stem cell transplantation; this was the case until introduction of the potent BCR/ABL tyrosine kinase inhibitor imatinib mesylate. IFN can activate the immune system to target and eradicate CML stem cells. A subset of HSCs is highly quiescent 40 . Thus, the effects of PARPi (i.e., creation of DSBs by inhibiting SSBs) may be limited in these cells. Activation of the cGAS/STING pathway may explain the effects of PARPi on BCR/ABL1-positive LSCs.
TKIs remain the gold standard treatment for CML and Ph1-ALL. A previous study shows that combination of a TKI with PARPi increases the antileukemic effect against BCR/ABL1-positive cells 27 . TKIs have markedly improved the outcome of patients with CML. However, only 40-60% of patients with CML that shows a deep

Material and methods
Cells and cell culture. KOPN30, BV173, and K562 are BCR/ABL1-positive leukemia cell lines. All leukemia cell lines, as well as Ba/F3 cells, were maintained in RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/mL) at 37 °C in an atmosphere containing 5% CO 2 . KOPN30 cells were obtained from the University of Yamanashi School of Medicine (Yamanashi, Japan). BV173 and Ba/F3 cells were obtained from DSMZ (Braunschweig, Germany). K562 cells were obtained from the JCRB cell Bank (Osaka, Japan). Rat-1 cells were obtained from RIKEN cell bank (Tsukuba, Japan). All cell lines were tested for mycoplasma contamination. Rat-1 cells and the fibroblast line MRC5SV harboring a single integrated copy of DR-GFP (DR-GFP MRC5SV) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin (100 units/mL) at 37 °C in an atmosphere containing 5% CO 2 .
DR-GFP assay. The BCR/ABL1-expressing plasmid was constructed by subcloning BCR/ABL1into the MSCV plasmid. The DR-GFP assay was performed as previously described 9 . Briefly, mock or BCR/ABL1expressing plasmids were transiently transfected into single-copy DR-GFP-integrated MRC5SV cells using X-tremeGENE 9 (Roche, Basel, Switzerland). On the next day, cells were transfected with the I-SceI expression vector pCBAS. GFP expression was monitored by flow cytometry 48 h after transfecting cells with pCBAS.
Cell death analyses. The percentage of apoptotic cells was measured by flow cytometry after staining with a combination of Annexin V (Abcam, Cambridge, MA) and propidium iodide 10 . Growth-inhibitory effects were assessed using a Cell Counting Kit (Dojindo, Kumamoto, Japan). A combination index, used to assess synergistic effects, was calculated using CompuSyn software 11 .

Rat-1 cell transformation assay.
A BCR/ABL1-mediated transformation assay using Rat-1 cells was performed as previously described 12 .Colony number was counted on day 21. Colony-forming activity was also measured using a CytoSelect™ 96 well Cell Transformation Assay Kit (Cell Biolabs, San Diego, CA).
Breakage-fusion-bridge (BFB) formation assay. Detection of nucleoplasmic bridges was used to assess BFB frequency; the assay was optimized for mouse cells as described previously 13 . Briefly, mouse HSCs were isolated using immunomagnetic columns as described above (Miltenyi Biotech) and then cultured in αMEM supplemented with 20% FCS, 50 ng/mL mouse SCF, 50 ng/mL mouse FLT3 ligand, 50 ng/mL human IL-6, and 50 ng/mL human TPO. Next, cells were exposed to 2 Gy X-ray irradiation and cultured for 48 h, followed by addition of cytochalasin-D (0.6 μg/mL) for 24 h. Then, cells were released from cytochalasin-D treatment for 2 h and exposed to cold hypotonic (0.075 M KCl) solution. Finally, cells were fixed in Carnoy fluid, dropped onto slides, stained with DAPI, and examined using a fluorescent microscope at a magnification of × 400.
Immunoprecipitation and western blotting. Cells  Hematopoietic stem cell (HSC) transplantation and transduction of BCR/ABL1. The BCR/ ABL1-expressing plasmid was constructed by subcloning BCR/ABL1 into the MSCV-IRES-GFP plasmid. Plat-E cells 20 , an ecotropic packaging cell line, were transfected with MSCV-BCR/ABL1-IRES-GFP using polyethyleneimine. Supernatants containing high titers of retrovirus were collected at 48 and 72 h and concentrated using a Retro-X Concentrator (TAKARA-Clontech, Ohtsu, Japan). LTR-HSCs were cultured overnight in αMEM supplemented with 20% FBS plus 50 ng/mL each of mouse stem cell factor (SCF), human IL-6, human FLT3 ligand, and human thrombopoietin (TPO). On Day 2, cells were placed in 24-well dishes coated with RetroNectin (TAKARA-Clontech, Shiga, Japan) and infected with concentrated retrovirus particles. At 60 h postinfection, retrovirus-infected LTR-HSCs were transplanted into mice that had received (6 h earlier) myeloablative conditioning with 9.5 Gy total body irradiation. Mice were allowed access (ad libitum) to water containing 1 mg/mL neomycin trisulfate salt hydrate and 100 U/mL polymyxin B sulfate salt.
Statistical analysis. P-values for the DR-GFP, apoptosis, cell survival, transformation, leukemic stem cell detection, and qPCR assays were calculated using a t test. Survival curves were constructed using the Kaplan-Meier method and analyzed using the log-rank test. All statistical tests were two-sided, and a p-value of < 0.05 was considered significant.